EP1771468B1 - Auf tumore zielende zellen, die den "tumor necrosis factor-related apoptosis-inducing ligand" (trial) produzieren - Google Patents

Auf tumore zielende zellen, die den "tumor necrosis factor-related apoptosis-inducing ligand" (trial) produzieren Download PDF

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EP1771468B1
EP1771468B1 EP05764208A EP05764208A EP1771468B1 EP 1771468 B1 EP1771468 B1 EP 1771468B1 EP 05764208 A EP05764208 A EP 05764208A EP 05764208 A EP05764208 A EP 05764208A EP 1771468 B1 EP1771468 B1 EP 1771468B1
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cells
trail
tumor
cell
apoptosis
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EP1771468A1 (de
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Alessandro M. Gianni
Carmelo Carlo-Stella
Francesco Colotta
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Dompe SpA
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Definitions

  • the present invention relates to tumor-homing cells expressing the tumor necrosis factor-related apoptosis inducing ligand (TRAIL) and the use thereof in anti-tumor therapy.
  • TRAIL tumor necrosis factor-related apoptosis inducing ligand
  • apoptotic pathways represent attractive therapeutic targets for restoring apoptosis sensitivity of malignant cells or activating agonists of apoptosis [2].
  • DR4, DR5, FLIP extrinsic
  • cIAP2 convergence
  • Tumor necrosis factor-related apoptosis-inducing ligand belongs to the TNF family of death receptor ligands [4].
  • Isolated DNA sequences coding for TRAIL, expression vectors comprising said DNA sequences and recombinant TRAIL polypeptides are disclosed in WO 97/01633 .
  • TRAIL binds to death receptor 4 (DR4) and death receptor 5 (DR5), which are expressed on the cell surface of many cancer cells [5]. Binding soluble TRAIL to DR4 or DR5 leads to to caspase activation and apoptosis [6].
  • TRAIL soluble recombinant TRAIL
  • TRAIL selectively induces apoptosis in many transformed cells, while sparing normal cells [4].
  • TRAIL administration in mice exerts a remarkable efficacy against tumor xenografts of colon carcinoma [8, 9], breast cancer [10], multiple myeloma [11] or glioma [12, 13].
  • soluble TRAIL An additional limitation to the use of soluble TRAIL is represented by the impaired apoptotic response observed in a substantial number and variety of tumor cell lines which require either a prolonged incubation time or very high dose of soluble TRAIL to undergo apoptosis [17, 18]. Given the pharmacokinetic profile of TRAIL after intravenous injection (plasmatic half-life of 32 minutes and elimination half-life of 4.8 hours), conditions of prolonged exposure time and high concentrations are not transferable for any in vivo treatment strategy [10]. In order to overcome limitations related to soluble recombinant TRAIL and specifically target tumor cells, several adenoviral-mediated TRAIL gene transfer approaches are currently being exploited using different animal models [19, 20].
  • adenoviral gene transfer approaches largely depend on the efficient infection of the tumor and avoidance of immune clearance to be effective [21].
  • several safety issues concerning the systemic injection of adenoviral vectors still remain to be addressed [22].
  • adenoviral gene transfer of TRAIL overcomes an impaired apoptotic response of hepatoma cells, but causes severe apoptosis in primary human hepatocytes [19].
  • adenoviral-based gene therapy approaches mainly rely on the intratumoral delivery of TRAIL-encoding adenovirus [23]. Despite a local antitumoral activity, intratumoral delivery of TRAIL has no systemic antitumor activity, thus lacking any clinical value.
  • CD34+ cells [25] and natural killer (NK) cells [26] may be used as delivery vehicles for anticancer molecules.
  • CD34+ cells display specific homing properties, including the capacity to permanently colonize the bone marrow, and transiently colonize the liver and spleen [27-30]. These homing properties are strictly related to the expression of adhesion receptors (e.g., CXCR-4, VLA-4, VLA-5, CD44, etc.) which interact with specific ligands (e.g., SDF-1, VCAM-1, etc.) which are expressed on stromal cells residing within the bone marrow microenvironment as well as the tumor microenvironment [31-34].
  • adhesion receptors e.g., CXCR-4, VLA-4, VLA-5, CD44, etc.
  • specific ligands e.g., SDF-1, VCAM-1, etc.
  • NK cells are a subset of lymphocytes that play an essential role in the cellular-based immune defense against tumor cells through major histocompatibility complex non-restricted mechanisms [35]. Following intravenous infusion, NK cells home to the bone marrow and lymphoid organs and extravasate at tumor sites under the influence of appropriate cytokine signals. Many groups have, therefore, sought to use NK cells to home to tumors for therapeutic purposes [36-38].
  • NK-cells Even though the tumor-homing properties of NK-cells is known, genetically modified cells undergo a stress which depend on the kind of the transduced gene. As a consequence of said stress, the cells may loose the original tumor-homing properties when transduced with the TRIAL gene. No reasonable expectation of success is therefore present and the actual experimental tests are necessary to confirm whether the specific considered approach may be viable or not.
  • CD34+ cells cells engineered to express TRAIL can be advantageously exploited for achieving a cell-mediated, anti-tumor activity in vivo.
  • CD34+ tumor-homing cells are engineered to produce tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) by adenoviral-mediated gene transfer.
  • TRAIL tumor necrosis factor-related apoptosis-inducing ligand
  • the cells according to the present invention can be systemically administered to deliver TRAIL to the tumor site without causing the aforementioned drawbacks.
  • the present invention relates to tumor-homing cells expressing TRAIL and cell preparations containing them.
  • the invention also relates to the use of said cells for the preparation of cell compositions for anti-cancer therapy, in particular for the therapy of human lymphoma.
  • the cells of the invention can be obtained by transducing tumor-homing cells with a replication-deficient adenovirus encoding the human TRAIL gene (Ad-TRAIL) under the control of a suitable promoter, e.g. the CMV promoter.
  • Ad-TRAIL a replication-deficient adenovirus encoding the human TRAIL gene
  • the transduction can be carried out according to methods well-known to molecular biologists, preferably following the procedure disclosed in the examples.
  • tumor-homing cells is used according to the invention to define cells able: (1) to home to tumor tissue following intravenous injection, and (2) to express adequate amounts of membrane-bound TRAIL (mTRAIL) for at least a few days.
  • mTRAIL membrane-bound TRAIL
  • the tumor-homing cells of the present invention are CD34+ hematopoietic cells from the peripheral blood of growth-factor treated cancer patients engineered to express mTRAIL.
  • mTRAIL Several methods can be envisaged in order to obtain adequate expression of mTRAIL, including use of plasmids and viral vectors with appropriate regulatory genetic elements such as tissue-specific promoters and/or enhancers.
  • An optimal transduction efficiency of CD34+ cells may be obtained exposing to graded (50 to 500) Multiplicity of Infection (MOI) of Ad-TRAIL (50 to 500) under serum-free conditions at 37°C. Serum-supplemented medium (RPMI-1640/FBS 20%) is then added, and a few hours later cultures were supplemented with Gene Booster (1:200) and further incubated for 18 hours.
  • MOI Multiplicity of Infection
  • compositions of the invention can be prepared by using vehicles suitable for parenteral, particularly intravenous, administration, such as those reported in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 1991 ).
  • excipients which may be used include any pharmaceutical agent that is not harmful to the individual receiving the composition, e.g. water, saline, glycerol and ethanol optionally in admixture with auxiliary substances, such as wetting or emulsifying agents, buffers, and the like in.
  • auxiliary substances such as wetting or emulsifying agents, buffers, and the like in.
  • Appropriate doses will depend, among other factors, on the sex, age and conditions of the subject to be treated, the severity of the disease. An appropriate effective amount can be readily determined by any skilled practitioner and may anyhow be determined by means of clinical studies.
  • a therapeutically effective dose will generally be from about 10 3 to about 10 15 of transduced cells. Other dosages can be of course established by means of conventional experiments, i.e.
  • Dosage treatment may be a single dose or a multiple dose schedule.
  • the subject may be administered as many doses as appropriate. The skilled practitioner will easily determine the most effective dosage regimen.
  • the effectiveness of intravenous administration provides a distinct advantage of the present invention in comparison protocols based to intra-tumor delivery.
  • Example 1 In vitro triggering of apoptosis in lymphoma cell lines co-cultured with CD34+ cells expressing TRAIL following adenoviral-mediated gene transfer
  • Ad-TRAIL adenoviral vector expressing TRAIL
  • Adenovirus encoding the human TRAIL gene A replication-deficient adenovirus encoding the human TRAIL gene (Ad-TRAIL) expressed from the CMV promoter was used for these experiments [39].
  • the Ad-TRAIL contains the entire coding sequence of human TRAIL cloned into the Xho I and Not I sites of pAd5CMVK-NpA.
  • the resultant plasmid and adenovirus backbone sequences (Ad5) that had the E1 genes deleted are transfected into human embryonic kidney 293 cells, and viral particles are isolated and amplified for analysis of TRAIL expression.
  • Recombinant adenoviruses are screened for replication competent virus by A549 plaque assay, and virus titer is determined by plaque assay on 293 cells. Purified viruses are stored in PBS with 3% sucrose and kept at -80°C until use. TRAIL gene product is expressed on the cell surface of transduced cells and can be detected by means of flow cytometry.
  • CD34+ cells to be transduced with Ad-TRAIL were obtained from the peripheral blood of consenting cancer patients receiving chemotherapy and treated with hematopoietic growth factors.
  • CD34+ cells were enriched from leukapheretic samples by means of an immunomagnetic technique and positive selection (Miltenyi Biotech).
  • IMDM serum-free medium
  • MOI multiplicity of infections
  • Co-cultures The apoptosis triggering activity of TRAIL expressed on the membrane of CD34+ cells was tested in vitro by means of co-culture experiments. Briefly, Ad-TRAIL/CD34+ cells (effector cells) were co-cultured with lymphoma cells (target cells). Apoptosis induction was evaluated 24 and 48 hours after the onset of co-culture. In these experiments, the effector: target cell ratio was calculated on the basis of the transduction efficiency of effector cells.
  • CD34+ cells Adenoviral transduction of CD34+ cells.
  • IMDM/FBS 20% An equal volume of serum-supplemented medium (IMDM/FBS 20%) was then added, and 4 hours later cultures were supplemented with Gene Booster (1:200) and further incubated for 18 hours.
  • Gene Booster Gene Booster
  • CD34+ cells were transduced with an MOI of 1,000 and analyzed for TRAIL expression up to 7 days after transduction.
  • MOI granulocyte colony-stimulating factor
  • G-CSF granulocyte colony-stimulating factor
  • CD34+ cells transduced under optimal conditions according to the infection protocols described above were collected 24 hours after the initial exposure to adenovirus, extensively washed and co-cultured with KMS-11 or JVM-2 cells.
  • Ad-TRAIL/CD34+ cells were co-cultured at an effector: target cell ratio of 0.8:1, a substantial proportion (81%) of apoptotic and necrotic cells was detected for KMS-11 cells after a 24-hour co-culture. The amount of apoptotic cells was further increased after a 48-hour co-culture (93% apoptotic cells).
  • Co-culture experiments were also performed by incubating Ad-TRAIL/CD34+ cells or Ad-TRAIL/NK cells with the soluble TRAIL-resistant NM-2 cell line.
  • Ad-TRAIL/CD34+ cells were co-cultured at an effector: target cell ratio of 0.8:1, a substantial proportion (51%) of apoptotic and necrotic cells was detected for the TRAIL-resistant JVM-2 cells after a 48-hour co-culture.
  • the potency of the apoptotic effect exerted by membrane-bound TRAIL was significantly higher than that exerted by a 48-hour exposure to a high-dose (100 ng/ml) of soluble TRAIL (table 4).
  • Table 4 - Effect of soluble TRAIL on JVM-2 cells Amount of soluble TRAIL % of viable cells after 24 hours % of viable cells after 48 hours 0 ng/ml 87 84 100 ng/ml 86 80
  • effector cells Ad-TRAIL/CD34+ cells were co-cultured with target cells (KMS-11 cell line) for 24 hours at different effector: target ratios.
  • Ad-TRAIL/CD34+ cells triggered a substantial percentage (66%) of KMS-11 cells to undergo apoptosis or necrosis in co-cultures set-up with an effector: target ratio of 0.4:1.
  • Table 5 - Induction of apoptosis by co-culturing Ad-TRAIL/CD34+ cells and KMS-11 cells for 24 hours at increasing E:T ratios CD34+/KMS-11 ratio 0:1 0.08:1 0.4:1 0.8:1 % of residual viable cells 71 67 34 22
  • Example 2 In vivo evaluation in NOD/SCID mice of the anticancer activity of CD34+ cells engineered to express TRAIL following adenoviral-mediated gene transfer
  • mice were reinfused with the TRAIL-sensitive KMS-11 multiple myeloma cell line. Subsequently, mice were injected with Ad-TRAIL/CD34+ cells and mice survival was used as a readout of the antitumor efficacy of cell-based TRAIL delivery.
  • mice were inoculated intravenously (IV) with KMS-11 cells (0.5 x 10 6 per mouse).
  • Treatment with Ad-TRAIL/CD34+ cells (1 x 10 6 per mouse) consisted in IV weekly injections for 4 weeks starting either on day 7 or 17 after tumor cell inoculation.
  • the mean transduction efficiencies of reinfused Ad-TRAIL/CD34+ cells were 83 ⁇ 8% and 61 ⁇ 18%, respectively.
  • each mouse received an average number of 3.32 x 10 6 TRAIL-expressing CD34+ cells over four injections.
  • Mice were checked twice weekly for tumor appearance, body weight measurements and toxicity. The survival times of animals in each group were recorded and differences among the groups were evaluated by statistical analysis.
  • Appropriate control groups included: (i) mice injected with tumor cells only, (iii) mice injected with tumor cells plus non-transduced CD34+ cells.
  • Ad-TRAIL/CD34+ cells Ad-TRAIL/CD34+ cells .
  • Mice xenografted with KMS-11 cells 0.5 x 10 6 per mouse
  • Example 3 In vitro antitumor activity of CD34/Ad-TRAIL cells against breast cancer cell lines
  • the sensitivity of breast cancer cell lines to the killing effects of sTRAIL was evaluated in comparison with the evaluation of the in vitro triggering of apoptosis of sTRAIL-sensitive and sTRAIL-resistant breast cancer cell lines co-cultured with CD34/Ad-TRAIL cells.
  • MCF-7 and MDA-MB-361 Two breast cancer cell lines, i.e., MCF-7 and MDA-MB-361were used.
  • tumor cells 5 - 10 x 10 4 /ml
  • a high (100 ng/ml) dose of sTRAIL were exposed for 72 hours to a low (10 ng/ml) and a high (100 ng/ml) dose of sTRAIL.
  • apoptosis was evaluated by annexin-V/propidium iodide double staining.
  • The-breast-cancer cell lines MCF-7 and MDA-MB-361 were purchased from the DSMZ (Braunschweig, Germany, EU) and ATCC (Manassas, VA, USA), respectively. Cells were periodically tested by polymerase chain reaction for mycoplasma contamination. All in vitro experiments were performed with exponentially growing cells.
  • Annexin-V expression The Annexin V-FITC assay (PharMingen) was used to quantitatively determine the percentage of cells undergoing early or late apoptosis and necrosis. Briefly, cells to be analyzed were washed twice with cold PBS and then resuspended in binding buffer (10 nM HEPES, 140 nM NaCI, 5 nM CaC12, pH 7.4). Following incubation, 0.1 ml of the cell suspension was transferred to a 5 ml culture tube and 5 microl of Annexin V-FITC and 10 microL of propidium iodide was added. After vortexing, the samples were incubated for 1 S min at room temperature in the dark. At the end of the incubation, 0.4 ml of binding buffer were added and the cells were analyzed immediately by flow cytometry.
  • MCF-7 and MDA-MB-361 cell lines were exposed to sTRAIL (10 - 100 ng/ml, 72 hours) and then the percentages of apoptotic and necrotic cells was detected by FACS analysis. As shown in Table 7, exposure to sTRAIL failed to induce any apoptotic response in MCF-7 cells, whereas it resulted in a significant cell death response by MDA-MB-361 cells when exposed to 100- ng/ml of sTRAIL for 72 hours. According to these results, MCF-7 is a sTRAIL-resistant cells line, whereas MDA-MB-361 is a sTRAIL-sensitive cell line.
  • Adenovirus encoding the human TRAIL gene A replication-deficient adenovirus encoding the human TRAIL gene (Ad-TRAIL) expressed from the CMV promoter was used for these experiments [39].
  • the Ad-TRAIL contains the entire coding sequence of human TRAIL cloned into the Xho I and Not I sites of pAd5CMVK-NpA.
  • the resultant plasmid and adenovirus backbone sequences (Ad5) that had the E1 genes deleted are transfected into human embryonic kidney 293 cells, and viral particles are isolated and amplified for analysis of TRAIL expression.
  • Recombinant adenoviruses are screened for replication competent virus by A549 plaque assay, and virus titer is determined by plaque assay on 293 cells. Purified viruses are stored in PBS with 3% sucrose and kept at -80°C until use. TRAIL gene product is expressed on the cell surface of transduced cells and can be detected by means of flow cytometry.
  • CD34+ cells to be transduced with Ad-TRAIL were obtained from the peripheral blood of consenting cancer patients receiving chemotherapy and treated with hematopoietic growth factors.
  • CD34+ cells were enriched from leukapheretic samples by means of an immunomagnetic technique and positive selection (Miltenyi Biotech).
  • IMDM serum-free medium
  • MOI multiplicity of infections
  • CD34+ cells Adenoviral transduction of CD34+ cells. An optimal transduction efficiency of CD34+ cells was consistently achieved by exposing CD34+ cells (2 x 10 6 /ml) to graded Ad-TRAIL at an MOI of 500 under serum-free conditions for 2 hours (37 °C). While no background TRAIL signal was detected in control cells, TRAIL-expressing cells revealed a percentage of TRAIL-positive CD34+ cells of 93 ⁇ 8% (mean ⁇ SD). Cell viability, as evaluated by the Trypan blue dye exclusion test, was unaffected by an MOI as high as 1,000 (with ⁇ 85% viable cells).
  • the potency of the apoptotic effect exerted by a 48-hour exposure to mTRAIL was similar to that exerted by a 72-hour exposure to a high-dose (100 ng/ml) of sTRAIL (Table 7).
  • MDA-MB-361 cells were co-cultured with mock-transduced CD34+ cells. As shown in Table 2, co-culturing MDA-MB-361 cells with mock-transduced CD34+ cells was associated with a modest cell death effect only detected at the highest E:T ratio. Such a modest cell death induction was likely related to culture overcrowding.
  • MDA-MB-361 cells were exposed to 10 6 plaque forming unit (pfu). No evidence of cell toxicity could be detected by exposing MDA-MB-361 cells to 10 6 viral particles (Table 8).
  • the number of pfu was calculated as follows.
  • CD34+ cells were washed 3x in culture medium.
  • a 1:20 dilution factor was used, i.e., the suspension culture was diluted at least 8,000-fold.
  • the potency of the apoptotic effect exerted by a 48-hour exposure to mTRAIL was significantly higher than that exerted by a 72-hour exposure to a high-dose (100 ng/ml) of sTRAIL (Tables 7 & 8).
  • MCF-7 cells either co-cultured with mock-transduced CD34+ cells. As shown in Table 8, co-culturing MCF-7 cells with mock-transduced CD34+ cells was associated with a modest cell death effect which is likely related to culture overcrowding.
  • MCF-7 cells were exposed to 10 6 pfu. No evidence of cell toxicity could be detected by exposing MCF-7 cells to 10 6 pfu (Table 8).

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Claims (7)

  1. CD34+ blutbildende Zellen, die aus peripherischem Blut von wachstumsfaktorbehandelten Krebspatienten stammen und mit dem Tumor-Nekrosis-Faktor-spezifischen Apoptose-induzierenden Ligand transduziert worden sind, mit der Bedingung, daß die Zellen keine dendritischen Zellen sind.
  2. Zellen nach Anspruch 1, welche durch Transduzieren von CD34+-Zellen mit adenoviralen Vektoren, welche für den Tumor-Nekrosis-Faktor-spezifischen Apoptose-induzierenden Ligand codieren, erzeugbar sind.
  3. Zellzusammensetzungen enthaltend die Zellen nach Ansprüchen 1-2 zusammen mit physiologisch zulässigen Trägerstoffen.
  4. Verwendung der Zellen nach Ansprüchen 1-2 für die Herstellung eines Arzneimittels für die Behandlung von Tumoren.
  5. Verwendung nach Anspruch 4, wobei der Tumor ein Lymphom ist.
  6. Verwendung nach Anspruch 4, wobei der Tumor ein Brustkarzinom ist.
  7. Verwendung nach einem der Ansprüche 4, 5 oder 6, wobei das Arzneimittel intravenös verabreicht wird.
EP05764208A 2004-07-29 2005-07-21 Auf tumore zielende zellen, die den "tumor necrosis factor-related apoptosis-inducing ligand" (trial) produzieren Active EP1771468B1 (de)

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PL05764208T PL1771468T3 (pl) 2004-07-29 2005-07-21 Zasiedlające nowotwory komórki inżynierowane w celu wytwarzania ligandu czynnika martwicy nowotworu indukującego apoptozę (TRAIL)
SI200530969T SI1771468T1 (sl) 2004-07-29 2005-07-21 CELICE, KI CILJAJO NA TUMORJE IN SO VZGOJENE ZA PROIZVODNJO "TUMOR NECROSIS FACTOR-RELATED APOPTOSIS-INDUCING LIGAND" (TRAIL)
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PCT/EP2005/007957 WO2006010558A1 (en) 2004-07-29 2005-07-21 Tumor-homing cells engineered to produce tumor necrosis factor-related apoptosis-inducing ligand (trail)
EP05764208A EP1771468B1 (de) 2004-07-29 2005-07-21 Auf tumore zielende zellen, die den "tumor necrosis factor-related apoptosis-inducing ligand" (trial) produzieren

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DE102006020307A1 (de) * 2006-05-03 2007-11-08 Martin-Luther-Universität Halle-Wittenberg TNF-related apoptosis-including (TRAIL)stabil transgen exprimierende mesenchymale Stammzellen, Verfahren zu ihrer Herstellung und zu ihrer Verwendung
WO2008142862A1 (ja) * 2007-05-18 2008-11-27 National University Corporation Asahikawa Medical College 血管内皮前駆細胞の移植による抗がん療法
CN102154213B (zh) * 2011-01-19 2012-07-25 郑骏年 一种运载荷载细胞因子的双调控溶瘤腺病毒的新型cik细胞
EP2811023B1 (de) * 2012-02-01 2018-10-24 Postech Academy-Industry Foundation Vektor zur gleichzeitigen expression von dodecamerischen pfad- und hsv-tk-selbstmordgenen sowie stammzellentherapeutikum damit
CN103288966B (zh) * 2013-05-17 2015-01-21 华侨大学 一种融合受体及其用于治疗大肠癌的基因药物
RU2552609C1 (ru) * 2013-10-28 2015-06-10 Федеральное государственное бюджетное учреждение науки Институт молекулярной биотехнологии им. В.А. Энгельгардта Российской академии наук (ИМБ РАН) Способ получения системы направленной доставки белковых молекул (онколитических белков) в опухолевые ткани на основе активированных лимфоцитов
EP3209382B1 (de) 2014-10-24 2020-11-25 Calidi Biotherapeutics, Inc. Kombinationsimmuntherapieansatz zur behandlung von krebs
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RS51381B (en) 2011-02-28
PL1771468T3 (pl) 2010-07-30
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AU2005266543A1 (en) 2006-02-02
CN101076540B (zh) 2012-10-24
RU2390558C2 (ru) 2010-05-27
EP1771468A1 (de) 2007-04-11
NO20070956L (no) 2007-02-20
IL180233A (en) 2010-12-30
DE602005019050D1 (de) 2010-03-11
CA2571426A1 (en) 2006-02-02
AU2005266543B2 (en) 2012-02-02
RU2007107369A (ru) 2008-09-10
DK1771468T3 (da) 2010-05-25
MX2007001152A (es) 2007-04-18
BRPI0513855A (pt) 2008-05-20
JP2008507961A (ja) 2008-03-21
PT1771468E (pt) 2010-04-20
HRP20100227T1 (hr) 2010-07-31
ZA200701231B (en) 2008-08-27
CN101076540A (zh) 2007-11-21
ATE455847T1 (de) 2010-02-15
IL180233A0 (en) 2007-07-04
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